Understanding pro-neuropeptide hormone processing, which can lead to the discovery of novel peptides with biologic function, and the way these products are delivered outside the cell is paramount to the fundamental role that these molecules play in animal physiology that relates to communication, and neuroendocrine function. ProThyrotropin Releasing Hormone (proTRH) is the protein precursor to TRH and other non-TRH peptides. TRH, produced in the paraventricular nucleus of the hypothalamus PVN, stimulates the biosynthesis and secretion of thyroid-stimulating hormone from the anterior pituitary. TSH, in turn, stimulates thyroid hormone biosynthesis and release. TRH is central in regulating the hypothalamic-pituitary thyroid (HPT) axis. TRH also plays a key role in maintaining energy balance and during cold stress by increasing the basic metabolic rate of the cells through the HPT axis. Therefore, in this proposal we will test the hypothesis that the sorting of the TRH prohormone to the regulated secretory pathway (RSP) involves one initial post-translational processing step in the Golgi complex by the prohormone convertase 1 (PC1), followed by two different pathways of sorting for each intermediate product of processing. These intermediate forms are then directed to the RSP by two potential sequence motifs: two RGDs (Arg-Gly-Asp) in the N-terminal intermediate and a potential disulfide bond structure formed at the C-terminal intermediate. Therefore, we will transiently transfect AtT-20 cells, and by comparison with the neuronal GT-1 cell line to prove the following hypotheses: Aim 1. The sorting of proTRH to the RSP requires initial PC activity that is governed by the secondary structure of proTRH. Aim 2. The sorting of the N- and C-terminal intermediate proTRH forms are directed by the RGD and a possible disulfide motifs respectively Aim 3. The sorting of the proTRH N- and C-terminal fragments is routed to two different pathways. Aim 4. The tertiary structure of proTRH contributes to its processing by PC1 and PC2 and further sorting to the RSP.